This invention relates to a composition of matter which comprises an antibody that is specifically reactive with a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1). The antibody includes monoclonal antibodies (MAbs), immunoreactive fragments or recombinants thereof. The invention also relates to pharmaceutical and diagnostic compositions and methods utilizing these antibodies.
In the developing embryo, the primary vascular network is established by in situ differentiation of mesodermal cells in a process called vasculogenesis. It is believed that all subsequent processes involving the generation of new vessels in the embryo and neovascularization in adults, are governed by the sprouting or splitting of new capillaries from the pre-existing vasculature in a process called angiogenesis (Pepper et al., Enzyme and Protein, 49: 138-162, 1996; Breier et al., Dev. Dyn., 204: 228-239, 1995; Risau, Nature, 386: 671-674, 1997). Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.
On the other hand, promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans.
The angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. The molecular mechanisms underlying the complex angiogenic processes are far from being understood.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGFxcex1), and hepatocyte growth factor (HGF). See for example Folkman et al., J. Biol. Chem., 267: 10931-10934, 1992 for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors, the vascular endothelial growth factors (VEGFs), and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF family, and appear to act primarily via endothelial receptor tyrosine kinases (RTKs).
Nine different proteins have been identified in the PDGF family, namely two PDGFs (A and B), VEGF and six members that are closely related to VEGF. The six members closely related to VEGF are: VEGF-B, described in International Patent Application PCT/US96/02957 (WO 96/26736) and in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki; VEGF-C, described in Joukov et al., EMBO J., 15: 290-298, 1996 and Lee et al., Proc. Natl. Acad. Sci. USA, 93: 1988-1992, 1996; VEGF-D, described in International Patent Application No.
PCT/US97/14696 (WO 98/07832), and Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-553, 1998; the placenta growth factor (PlGF), described in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267-9271, 1991; VEGF2, described in International Patent Application No. PCT/US94/05291 (WO 95/24473) by Human Genome Sciences, Inc; and VEGF3, described in International Patent Application No. PCT/US95/07283 (WO 96/39421) by Human Genome Sciences, Inc. Each VEGF family member has between 30% and 45% amino acid sequence identity with VEGF. The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cysteine knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity for endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life (Carmeliet et al., Nature, 380: 435-439, 1996; Ferrara et al., Nature, 380: 439-442, 1996; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 18: 4-25, 1997). The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele results in embryonic lethality due to failed development of the vasculature (Carmeliet et al., Nature, 380: 435-439, 1996; Ferrara et al., Nature, 380: 439-442, 1996). In addition VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF). The isolation and properties of VEGF have been reviewed; see Ferrara et al., J. Cellular Biochem., 47: 211-218, 1991 and Connolly, J. Cellular Biochem., 47: 219-223, 1991. Alterative mRNA splicing of a single VEGF gene gives rise to five isoforms of VEGF.
VEGF-B has similar angiogenic and other properties to those of VEGF, but is distributed and expressed in tissues differently from VEGF. In particular, VEGF-B is very strongly expressed in heart, and only weakly in lung, whereas the reverse is the case for VEGF. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, may have functional differences.
VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular resinoid acid-binding protein type I (CRABP-I). Its isolation and characteristics are described in detail in PCT/US96/02957 and in Olofsson et al., Proc. Natl. Acad. Sci. USA, 93: 2576-2581, 1996.
VEGF-C was isolated from conditioned media of the PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cells transfected to express VEGFR-3. VEGF-C was purified using affinity chromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al., EMBO J., 15: 290-298, 1996.
VEGF-D was isolated from a human breast cDNA library, commercially available from Clontech, by screening with an expressed sequence tag obtained from a human cDNA library designated xe2x80x9cSoares Breast 3NbHBstxe2x80x9d as a hybridization probe (Achen et al., Proc. Natl. Acad. Sci. USA, 95: 548-553, 1998). Its isolation and characteristics are described in detail in International Patent Application No. PCT/US97/14696 (WO98/07832).
The VEGF-D gene is broadly expressed in the adult human, but is certainly not ubiquitously expressed. VEGF-D is strongly expressed in heart, lung and skeletal muscle. Intermediate levels of VEGF-D are expressed in spleen, ovary, small intestine and colon, and a lower expression occurs in kidney, pancreas, thymus, prostate and testis. No VEGF-D mRNA was detected in RNA from brain, placenta, liver or peripheral blood leukocytes.
PlGF was isolated from a term placenta cDNA library. Its isolation and characteristics are described in detail in Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267-9271, 1991. Presently its biological function is not well understood.
VEGF2 was isolated from a highly tumorgenic, oestrogen-independent human breast cancer cell line. While this molecule is stated to have about 22% homology to PDGF and 30% homology to VEGF, the method of isolation of the gene encoding VEGF2 is unclear, and no characterization of the biological activity is disclosed.
VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is stated to have about 36% identity and 66% similarity to VEGF. The method of isolation of the gene encoding VEGF3 is unclear and no characterization of the biological activity is disclosed.
Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions.
PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases. Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), Tie and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The essential, specific role in vasculogenesis and angiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos.
The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C has been shown to be the ligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al., The EMBO Journal, 15: 290-298, 1996). VEGF-D binds to both VEGFR-2 and VEGFR-3. A ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified.
Recently, a novel 130-135 kDa VEGF isoform specific receptor has been purified and cloned (Soker et al., Cell, 92: 735-745, 1998). The VEGF receptor was found to specifically bind the VEGF165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al., Cell, 92: 735-745, 1998). Surprisingly, the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al., J. Biol. Chem., 273: 22272-22278, 1998).
VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 8: 11-18, 1992; Kaipainen et al., J. Exp. Med., 178: 2077-2088, 1993; Dumont et al., Dev. Dyn., 203: 80-92, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 9: 3566-3570, 1995). VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation. Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., Nature, 376: 66-70, 1995). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 generates viable mice with a normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA, 95: 9349-9354, 1998). The reasons underlying these differences remain to be explained but suggest that receptor signalling via the tyrosine kinase is not required for the proper function of VEGFR-1. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., Nature, 376: 62-66, 1995; Shalaby et al., Cell, 89: 981-990, 1997). Inactivation of VEGFR-3 results in cardiovascular failure due to abnormal organization of the large vessels (Dumont et al. Science, 282: 946-949, 1998).
Although VEGFR-1 is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., Nature, 376: 66-70, 1995). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 87: 3336-3343, 1995). In embryos, VEGFR-1 is expressed by most, if not all, vessels (Breier et al., Dev. Dyn., 204: 228-239, 1995; Fong et al., Dev. Dyn., 207: 1-10, 1996).
The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 54: 6571-6577, 1994; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570, 1995). VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis. Targeted inactivation of both copies of the VEGFR-3 gene in mice resulted in defective blood vessel formation characterized by abnormally organized large vessels with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at post-coital day 9.5. On the basis of these findings it has been proposed that VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels. However, the role of VEGFR-3 in the development of the lymphatic vasculature could not be studied in these mice because the embryos died before the lymphatic system emerged. Nevertheless it is assumed that VEGFR-3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis. Furthermore this suggests that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., EMBO J., 15: 290-298, 1996).
Some inhibitors of the VEGF/VEGF-receptor system have been shown to prevent tumor growth via an anti-angiogenic mechanism; see Kim et al., Nature, 362: 841-844, 1993 and Saleh et al., Cancer Res., 56: 393-401, 1996.
The invention generally provides a composition of matter which comprises an antibody specifically reactive with a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1), where the antibody is a monoclonal antibody (MAb), an immunoreactive fragment or a recombinant thereof. In another embodiment of the invention, the composition of matter comprises an antibody that can interfere with the activity of VEGF-D mediated by the mammalian VEGFR-2 and/or interfere with the binding of VEGF-D to the mammalian VEGFR-3. A particularly preferred antibody can interfere with the activity of VEGF-D mediated by VEGFR-2 and/or interfere with the binding of VEGF-D to VEGFR-3 but not with the activity of VEGF mediated by VEGFR-2 and/or bind to VEGF-C. The invention also relates to pharmaceutical and diagnostic compositions and methods utilizing the antibody.
According to a first aspect, the invention provides for a composition of matter which comprises an antibody specifically reactive with the polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1). Examples of such antibodies include the monoclonal antibodies (MAbs) designated 2F8, 4A5, 4E10, 5F12, 4H4 and 3C10.
According to a second aspect, the invention provides a composition of matter which comprises an antibody which interferes with the activity of VEGF-D mediated by the VEGFR-2. In a preferred embodiment this antibody also interferes with the binding of VEGF-D to VEGFR-3 but does not interfere with the activity of VEGF mediated by the VEGFR-2 and/or bind to VEGF-C. An example of such an antibody is the antibody MAb 4A5 which has the isotype, IgG1.
In a third aspect, the invention provides a composition of matter which comprises an antibody which interferes with the binding of VEGF-D to VEGFR-3.
Antibodies can be raised against the polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ. ID NO:1) or a fragment of the polypeptide using standard methods in the art. In addition this polypeptide can be linked to an epitope tag, such as the FLAG(copyright) octapeptide (Sigma, St. Louis, Mo.), to assist in affinity purification. For some purposes, for example where an antibody is to be used to inhibit effects of VEGF-D in a clinical situation, it may be desirable to use humanized or chimeric monoclonal antibodies or immunoreactive fragments thereof. Methods for producing these are given below, and are also well known in the art, including recombinant DNA methods.
These aspects of the invention also include MAbs, immunoreactive fragments or recombinants thereof, and they may all be suitably labeled.
Antibodies according to the invention may be labeled with a detectable label and utilized for diagnostic purposes. The antibody may be covalently or non-covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, ecogenic or radioactive agent for imaging. For use in diagnostic assays, radioactive or non-radioactive labels may be used. Examples of radioactive labels include a radioactive atom or group, such as 125I or 32P. Examples of non-radioactive labels include enzymatic labels, such as horseradish peroxidase, or fluorimetric labels, such as fluorescein-5-isothiocyanate (FITC) . Labeling may be direct or indirect, covalent or non-covalent.
A fourth aspect of the invention relates to a method for preparing a monoclonal antibody that is specifically reactive with a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1). In addition this polypeptide can be linked to an epitope tag, such as the
FLAG(copyright) octapeptide(Sigma-Aldrich). The method comprises the steps of immunizing an immunocompetent mammal with an immunogen comprising a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) or a fragment of the polypeptide and, optionally, a linked epitope tag; fusing lymphocytes of the immunized immunocompetent mammal with myeloma cells to form hybridoma cells; screening monoclonal (MAbs) produced by the hybridoma cells for specific binding activity to the polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1); culturing a hybridoma cell producing MAbs having specific binding activity to the polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) in a medium to proliferate and/or to secrete said monoclonal antibody; and recovering said monoclonal antibody from the culture supernatant.
In addition, a method is provided for preparing a monoclonal antibody that interferes with the activity of VEGF-D mediated by VEGFR-2 and/or interferes with the binding of VEGF-D with VEGFR-3. This method comprises the steps of immunizing an immunocompetent mammal with an immunogen comprising a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) or a fragment of the polypeptide and, optionally, a linked epitope tag; fusing lymphocytes of the immunized immunocompetent mammal with myeloma cells to form hybridoma cells; screening MAbs produced by the hybridoma cells for VEGF-D interfering activity and/or VEGF-D binding interfering activity; culturing a hybridoma cell producing MAbs having VEGF-D interfering activity and/or VEGF-D binding interfering activity in a medium to proliferate and/or to secrete said monoclonal antibody; and recovering said monoclonal antibody from the culture supernatant. In both methods the preferred immunocompetent mammal is a mouse or a rat.
A fifth aspect of the invention is a hybridoma cell that produces a monoclonal antibody that is specifically reactive with a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) or that interferes with the activity of VEGF-D mediated by the VEGFR-2 and/or interferes with VEGF-D binding to VEGFR-3.
A sixth aspect of the invention provides a method for preparing a hybridoma that produces a monoclonal antibody that is specifically reactive with a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) and/or that interferes with the activity of VEGF-D mediated by the VEGFR-2 and/or interferes with VEGF-D binding to VEGFR-3 which comprises the steps of immunizing an immunocompetent mammal with an immunogen comprising a polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) or a fragment of the polypeptide and, optionally, a linked epitope tag; obtaining lymphocytes of the immunized immunocompetent mammal; fusing the lymphocytes with myeloma cells to form hybridoma cells; screening MAbs produced by the hybridoma cells for specific binding activity to the polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) and/or VEGF-D interfering activity and/or VEGF-D binding interfering activity; and culturing a hybridoma cell that produces said monoclonal antibody having specific binding activity to the polypeptide having the amino acid sequence as set forth in FIG. 1 (SEQ ID NO:1) and/or VEGF-D interfering activity and/or VEGF-D binding interfering activity.
The term xe2x80x9cculturingxe2x80x9d refers to the cloning of the hybridoma cell by causing it to proliferate and to the induction of the hybridoma to secrete the antibodies described above.
A further aspect of the invention provides a method of interfering with at least one biological activity induced by VEGF-D in a mammal or in a cell culture which comprises the step of administering to said mammal or adding to said cell culture an effective biological activity interfering amount of the monoclonal antibody.
The xe2x80x9cbiological activities induced by VEGF-Dxe2x80x9d can be readily tested by methods known in the art. In particular, VEGF-D has the ability to stimulate endothelial cell proliferation or differentiation, including, but not limited to, proliferation or differentiation of vascular endothelial cells and/or lymphatic endothelial cells. Other biological activities contemplated include angiogenesis, lymphangiogenesis and induction of permeability of blood vessels and lymphatic vessels.
The term xe2x80x9cantibodiesxe2x80x9d or xe2x80x9cantibodyxe2x80x9d refers to the composition of matter which comprises an antibody that can interfere with the activity of VEGF-D mediated by the mammalian VEGFR-2 and/or interfere with the binding of VEGF-D to the mammalian VEGFR-3. A particularly preferred antibody can interfere with the activity of VEGF-D mediated by VEGFR-2 and/or interfere with the binding of VEGF-D to VEGFR-3 but not with the activity of VEGF mediated by VEGFR-2 and/or bind to VEGF-C.
Another aspect of the invention concerns the provision of a pharmaceutical composition comprising a therapeutically effective amount of an antibody, and a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier or adjuvant. Examples of such a carrier or adjuvant include, but are not limited to, saline, buffered saline, Ringer""s solution, mineral oil, talc, corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, dextrose, water, glycerol, ethanol, thickeners, stabilizers, suspending agents and combinations thereof. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, elixirs, syrups, wafers, ointments or other conventional forms. The formulation to suit the mode of administration. Compositions comprising PDGF-C will contain from about 0.1% to 90% by weight of the active compound(s), and most generally from about 10% to 30%.
The dose(s) and route of administration will depend upon the nature of the patient and condition to be treated, and will be at the discretion of the attending physician or veterinarian. Suitable routes include oral, subcutaneous, intramuscular, intraperitoneal or intravenous injection, parenteral, topical application, implants etc. For example, an effective amount of an antibody is administered to an organism in need thereof in a dose between about 0.1 and 1000 xcexcg/kg body weight.
Clinical applications of the invention include diagnostic applications and suppression or inhibition of angiogenesis and/or lymphangiogenesis in the treatment of cancer, diabetic retinopathy, psoriasis and arthopathies. Thus the invention also relates to a method of interfering with angiogenesis, lymphagiogenesis and/or neovascularization in a mammal in need of such treatment which comprises the step of administering an effective amount of an antibody to the mammal. The antibody interferes with the action of VEGF-D by preventing the activation of at least VEGFR-2.
In addition, this aspect of the invention provides a method of interfering with at least one biological activity selected from angiogenesis, lymphangiogenesis and neovascularization in a disease in a mammal selected from the group of cancer, diabetic retinopathy, psoriasis and arthopathies, comprising the step of administering to said mammal an effective angiogenesis, lymphangiogenesis or neovascularization interfering amount of the antibody. As noted above, the antibody interferes with the action of VEGF-D at least in part by interfering with the activity of VEGF-D mediated by VEGFR-2 and/or with the binding of VEGF-D to VEGFR-3.
The antibodies can be used to treat conditions, such as congestive heart failure, involving accumulations of fluid in, for example, the lung resulting from increases in vascular permeability, by exerting an offsetting effect on vascular permeability in order to counteract the fluid accumulation. Accordingly, the invention provides a method for treating fluid accumulation in the heart and/or lung due to increases in vascular permeability in a mammal. This method comprises administering to said mammal in need of such treatment an effective vascular permeability decreasing amount of an antibody.
The invention also provides a method of detecting VEGF-D in a biological sample, comprising the step of contacting the sample with an antibody, and detecting binding involving the antibody. In a preferred embodiment the binding and/or extent of binding is detected by means of a detectable label; suitable labels are discussed above.
According to yet a further aspect, the invention provides a diagnostic/prognostic device typically in the form of a test kit. For example, in one embodiment of the invention there is provided a diagnostic/prognostic test kit comprising the antibody to the polypeptide of FIG. 1 (SEQ ID NO: 1) and means for detecting the binding between the antibody and VEGF-D. In one preferred embodiment of the diagnostic/prognostic device according to the invention, a second antibody (a secondary antibody) directed against antibodies of the same isotype and animal source of the antibody directed against polypeptide of FIG. 1 (SEQ ID NO:1) (the primary antibody) is provided. The secondary antibody is coupled to a detectable label and then either an unlabeled primary antibody or VEGF-D is substrate-bound so that the VEGF-D/primary antibody interaction can be established by determining the amount of label bound to the substrate following binding between the primary antibody and VEGF-D and the subsequent binding of the labeled secondary antibody to the primary antibody. In a particularly preferred embodiment of the invention, the diagnostic/prognostic device may be provided as a conventional enzyme-linked immunosorbent assay (ELISA) kit.
According to yet a further aspect, the invention provides a method for identifying a compound which interferes with the interaction between VEGFR-3 and VEGF-D. This method comprises applying a polypeptide having the extracellular domain of VEGFR-3 to a substrate, incubating the substrate with VEGF-D in the presence of the compound to be identified, and detecting any interaction between VEGFR-3 and VEGF-D.
According to yet a further aspect, the invention provides a method for imaging of lymphatic vasculature in tissue, which comprises contacting the tissue with an antibody, and detecting the occurrence of binding of the antibody.